涉案偶蹄目动物DNA条形码构建

刘大伟; 侯森林; 周用武; 费宜玲; 何建

doi:10.3969/j.issn.1000-2006.202004013

涉案偶蹄目动物DNA条形码构建

刘大伟, 侯森林, 周用武, 费宜玲, 何建

南京林业大学学报（自然科学版） ›› 2020, Vol. 44 ›› Issue (6) : 27-32.

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南京林业大学学报（自然科学版） ›› 2020, Vol. 44 ›› Issue (6) : 27-32. DOI: 10.3969/j.issn.1000-2006.202004013

专题报道（执行编委鲁长虎）

涉案偶蹄目动物DNA条形码构建

作者信息 +

DNA barcoding of Artiodactyla in cases involved in wildlife for species identification

Author information +

文章历史 +

摘要

【目的】利用线粒体DNA条形码标记鉴定涉案偶蹄目动物,为解决偶蹄目动物残体、制品的鉴定难题提供技术参考。【方法】对16个偶蹄目动物物种共67份样本,采用试剂盒提取DNA,扩增、测定物种的线粒体DNA的COI基因、Cyt b基因片段;在GenBank上Blast搜索进行同源性分析;利用MEGA 7.0软件分别比较这两个基因片段种内和种间遗传距离,构建系统发育树。【结果】在同源性分析中,西伯利亚狍(Capreolus pygargus)的Cyt b基因片段与GenBank中西伯利亚狍、欧洲狍(Capreolus capreolus)基因序列的同源性均为99.64%~99.82%,该片段不能区分西伯利亚狍与欧洲狍这两个近缘种。其余测序结果与数据库中参考序列一致且相似度较高。对于COI片段,种内遗传距离为0~1.4%,种间遗传距离为1.8%~18.8%。对于Cyt b基因片段种内遗传距离为0~0.5%,种间遗传距离为4.0%~15.8%。两个片段的种内遗传差异均小于种间遗传差异,存在较为明显的条形码间隔。COI和Cyt b基因片段的系统发育树中,同物种的不同个体均能形成具有较高支持度的单分支;不同物种之间界限清晰,每个物种的所有个体均能够形成具有较高支持度的单分支。【结论】基于COI和Cyt b基因片段构建的DNA条形码均可用于对涉案偶蹄目动物的物种鉴定。在物种鉴定中,可采用两个片段联合使用的方式,尤其是对于近缘种的鉴定,以降低错误风险。

Abstract

【Objective】In this study, we aimed to identify Artiodactyla species in cases of wildlife destruction, using mitochondrial DNA barcoding. 【Method】COI and Cyt b sequences in 67 samples from 16 species were amplified. Animal samples were identified using Blast search on the GenBank sequences. Intra-and inter-specific genetic distances were compared, and phylogenetic trees were constructed using MEGA 7.0. 【Result】In a homology analysis, the Cyt b sequences of Capreolus pygargus and Capreolus capreouls were 99.64%-99.82% homologous; this sequence could not distinguish two closely related species, whereas other COI and Cyt b sequences could. The intra-specific genetic distance was 0-1.4% for COI and 0-0.5% for Cyt b, whereas the inter-specific genetic distance was 1.8%-18.8% for COI and 4.0%-15.8% for Cyt b. An obvious barcoding gap was detected in both markers for all species. The phylogenetic tree showed that samples from the same species formed monophyletic groups with high support values. The gaps between different species were clear, and all samples from each species formed a single branch with a high support value.【Conclusion】COI and Cyt b may serve as molecular markers for Artiodactyla identification in cases involving wildlife resources. To minimize the risk of false identification, the two DNA barcodes could be used simultaneously, especially for the closely related species.

导出引用

刘大伟, 侯森林, 周用武, 等. 涉案偶蹄目动物DNA条形码构建[J]. 南京林业大学学报（自然科学版）. 2020, 44(6): 27-32 https://doi.org/10.3969/j.issn.1000-2006.202004013

LIU Dawei, HOU Senlin, ZHOU Yongwu, et al. DNA barcoding of Artiodactyla in cases involved in wildlife for species identification[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2020, 44(6): 27-32 https://doi.org/10.3969/j.issn.1000-2006.202004013

中图分类号： Q34;S718.6

参考文献

列表( 原文顺序 | 文献年度倒序 | 文中引用次数倒序 ) 可视化分析

[1]	HEBERT P D N, CYWINSKA A, BALL S L, et al. Biological identifications through DNA barcodes[J]. Proceedings of the Royal Society of London.Series B: Biological Sciences, 2003,270:313-321.DOI: 10.2307/3558697. 本文引用 [1]

[2]	杨倩倩, 刘苏汶, 俞晓平. DNA条形码分析方法研究进展[J]. 应用生态学报, 2018,29(3):1006-1014. YANG Q Q, LIU S W, YU X P. Research progress on DNA barcoding analysis methods[J]. Chin J Appl Ecol, 2018,29(3):1006-1014.DOI: 10.13287/j.1001-9332.201803.032. 本文引用 [1]

[3]	HOFMANN T, KNEBELSBERGER T, KLOPPMANN M, et al. Egg identification of three economical important fish species using DNA barcoding in comparison to a morphological determination[J]. J Appl Ichthyol, 2017,33(5):925-932.DOI: 10.1111/jai.13389. 本文引用 [1]

[4]	YU M, JIAO L C, GUO J, et al. DNA barcoding of vouchered xylarium wood specimens of nine endangered Dalbergia species[J]. Planta, 2017,246(6):1165-1176.DOI: 10.1007/s00425-017-2758-9. 本文引用 [1]

[5]

赵志鹏, 向余劲攻, 何鑫, 等.用mtDNA序列鉴定出上海海域须鲸科物种大村鲸[J]. 动物学杂志, 2019, 54(4):493-500.

ZHAO Z

, XIANGYU J

, HE

, et al. Identification of Balaenoptera omurai (Balaenopteridae) from Shanghai seas by sing mtDNA gene sequences[J]. Chinese Journal of Zoology, 2019,54(4):493-500.DOI: 10.13859/j.cjz.201904006.

本文引用 [1]

[6]	程希婷, 王爱民, 顾志峰, 等. DNA条形码研究进展[J]. 基因组学与应用生物学, 2011,30(6):748-758. CHENG X T, WANG A M, GU Z F, et al. Current progress of DNA barcoding[J]. Genom Appl Biol, 2011,30(6):748-758.DOI: 10.3969/gab.030.000748. 本文引用 [1]

[7]	RATNASINGHAM S, HEBERT P D N.BOLD: the barcode of life data system[J]. Molecular Ecology Notes, 2007,7:355-364.DOI: 10.1046/j.1365-2540.1998.00318.x. 本文引用 [1]

[8]	SHEARER T L, VAN OPPEN M J H, ROMANO S L, et al.Slow mitochondrial DNA sequence evolution in the Anthozoa (Cnidaria)[J]. Mol Ecol, 2002,11(12):2475-2487.DOI: 10.1046/j.1365-294x.2002.01652.x. 本文引用 [1]

[9]	梁刚, 张卫, 雷富民, 等. 雀形目15种鸟类COⅠ与Cytb基因序列的比较[J]. 动物分类学报, 2007,32(3):613-620. LIANG G, ZHANG W, LEI F M, et al. Comparison of cyt b and COI gene sequences from 15 species in Passeriformes[J]. Acta Zootaxonomica Sin, 2007,32(3):613-620.DOI: 10.3969/j.issn.1000-0739.2007.03.027. 本文引用 [1]

[10]	MCFADDEN C S, BENAYAHU Y, PANTE E, et al. Limitations of mitochondrial gene barcoding in Octocorallia[J]. Mol Ecol Resour, 2011,11(1):19-31.DOI: 10.1111/j.1755-0998.2010.02875.x. 本文引用 [1]

[11]	KOCHER T D, THOMAS W K, MEYER A, et al. Dynamics of mitochondrial DNA evolution in animals:amplification and sequencing with conserved primers[J]. PNAS, 1989,86(16):6196-6200.DOI: 10.1073/pnas.86.16.6196. 本文引用 [1]

[12]	JOHNSON T A, IYENGAR A. Phylogenetic evidence for a case of misleading rather than mislabeling in caviar in the United Kingdom[J]. J Forensic Sci, 2015,60:S248-S253.DOI: 10.1111/1556-4029.12583. 本文引用 [1]

[13]	李楠, 王佳慧, 韩春卉, 等. COI及Cyt b基因对河鲀鱼鱼种鉴定的适用性研究[J]. 中国食品卫生杂志, 2018,30(1):6-11. LI N, WANG J H, HAN C H, et al. Application of COI and Cyt b gene in species identification of pufferfish[J]. Chin J Food Hyg, 2018,30(1):6-11.DOI: 10.13590/j.cjfh.2018.01.002. 本文引用 [1]

[14]	KUMAR S, STECHER G, TAMURA K. MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets[J]. Mol Biol Evol, 2016,33(7):1870-1874.DOI: 10.1093/molbev/msw054. 本文引用 [1]

[15]	冯成利, 冯慧, 王艳. 陕西省非法贸易动物分布及现状[J]. 陕西农业科学, 2012,58(4):179-184. FENG C L, FENG H, WANG Y. Distribution of illegal trade animal in Shaanxi Province[J]. Shaanxi J Agric Sci, 2012,58(4):179-184.DOI: 10.3969/j.issn.0488-5368.2012.04.061. 本文引用 [1]

[16]	胡诗佳, 彭建军, 于冬梅, 等. 华南地区兽类野生动物非法贸易状况及分析[J]. 四川动物, 2011,30(2):293. HU S J, PENG J J, YU D M, et al. Analysis on the illegal trade of wild animals in South China[J]. Sichuan J Zool, 2011,30(2):293. 本文引用 [1]

[17]	熊小倩, 尤炜轩, 张玲, 等. 北京地区野生动物资源非法利用状况分析[J]. 野生动物学报, 2017,38(3):376-385. XIONG X Q, YOU W X, ZHANG L, et al.Status of illegal use of wildlife resources in Beijing[J]. Chin J Wildl, 2017,38(3):376-385.DOI: 10.19711/j.cnki.issn2310-1490.2017.03.006. 本文引用 [1]

[18]	ZENG Y, WU Z Z, ZHANG C G, et al. DNA barcoding of mobulid ray gill rakers for implementing CITES on elasmobranch in China[J]. Sci Rep, 2016,6(1):37567.DOI: 10.1038/srep37567. 本文引用 [1]

[19]

NGATIA J

, LAN T

, MA

, et al. Distinguishing extant elephants Ivory from mammoth ivory using a short sequence of cytochrome b gene[J]. Sci Rep, 2019,9:18863.DOI: 10.1038/s41598-019-55094-x.

https://doi.org/10.1038/s41598-019-55094-x

https://www.ncbi.nlm.nih.gov/pubmed/31827140

本文引用 [2] 摘要

Trade in ivory from extant elephant species namely Asian elephant (Elephas maximus), African savanna elephant (Loxodonta africana) and African forest elephant (Loxodonta cyclotis) is regulated internationally, while the trade in ivory from extinct species of Elephantidae, including woolly mammoth, is unregulated. This distinction creates opportunity for laundering and trading elephant ivory as mammoth ivory. The existing morphological and molecular genetics methods do not reliably distinguish the source of ivory items that lack clear identification characteristics or for which the quality of extracted DNA cannot support amplification of large gene fragments. We present a PCR-sequencing method based on 116 bp target sequence of the cytochrome b gene to specifically amplify elephantid DNA while simultaneously excluding non-elephantid species and ivory substitutes, and while avoiding contamination by human DNA. The partial Cytochrome b gene sequence enabled accurate association of ivory samples with their species of origin for all three extant elephants and from mammoth. The detection limit of the PCR system was as low as 10 copy numbers of target DNA. The amplification and sequencing success reached 96.7% for woolly mammoth ivory and 100% for African savanna elephant and African forest elephant ivory. This is the first validated method for distinguishing elephant from mammoth ivory and it provides forensic support for investigation of ivory laundering cases.

[20]	ROLO E A, OLIVEIRA A R, DOURADO C G, et al. Identification of sarcosaprophagous Diptera species through DNA barcoding in wildlife forensics[J]. Forensic Sci Int, 2013,228(1/2/3):160-164. DOI: 10.1016/j.forsciint.2013.02.038. 本文引用 [1]

[21]	秦啼, 王东权, 曲宁新, 等. 基于粪便DNA技术的鸟类物种鉴定体系研究[J]. 野生动物学报, 2020,41(1):145-151. QIN T, WANG D Q, QU N X, et al. Bird species identification using faecal DNA analysis[J]. Chin J Wildl, 2020,41(1):145-151.DOI: 10.3969/j.issn.1000-0127.2020.01.020. 本文引用 [1]

[22]

侯森林, 费宜玲, 赵国清, 等. 3种鹿科动物针毛扫描电镜观察[J]. 江苏林业科技, 2018,45(5):35-38.

HOU S

, FEI Y

, ZHAO G

, et al. A survey on the micro-structural morphological character of guard hair of 3 species of deer by SEM[J]. Journal of Jiangsu Forestry Science & Technology, 2018,45(5):35-38.DOI: 10.3969/j.issn.1001-7380.2018.05.008.

本文引用 [1]

[23]	KÖHLER F. From DNA taxonomy to barcoding-how a vague idea evolved into a biosystematic tool[J]. Zool Reihe, 2007,83(S1):44-51.DOI: 10.1002/mmnz.200600025. 本文引用 [1]

[24]	HEBERT P D N, RATNASINGHAM S, DEWAARD J R. Barcoding animal life:cytochrome c oxidase subunit 1 divergences among closely related species[J]. Proc Biol Sci, 2003,270(S 1):S96-S99.DOI: 10.1098/rsbl.2003.0025. 本文引用 [1]

[25]	SHEN Y J, GUAN L H, WANG D Q, et al. DNA barcoding and evaluation of genetic diversity in Cyprinidae fish in the midstream of the Yangtze River[J]. Ecol Evol, 2016,6(9):2702-2713.DOI: 10.1002/ece3.2060. 本文引用 [1]

[26]	王业磷, 章群, 邓春兴. 基于线粒体COI基因的马鲅科鱼类DNA条形码研究[J]. 海洋渔业, 2020,42(1):1-9. WANG Y L, ZHANG Q, DENG C X, et al. DNA barcoding of Polynemidae based on mitochondrial COI gene[J]. Marine Fisheries, 2020,42(1):1-9.DOI: 10.13233/j.cnki.mar.fish.2020.01.001. 本文引用 [2]

[27]

DINH T

, NGATIA J

, CUI L

, et al. Influence of pairwise genetic distance computation and reference sample size on the reliability of species identification using Cyt b and COI gene fragments in a group of native passerines[J]. Forensic Sci Int:Genet, 2019,40:85-95.DOI: 10.1016/j.fsigen.2019.02.013.

https://www.ncbi.nlm.nih.gov/pubmed/30780122

本文引用 [1]

[28]

JIANG

, JIN

, LIANG

, et al. Existence of species complex largely reduced barcoding success for invasive species of Tephritidae:a case study in Bactrocera spp.[J]. Mol Ecol Resour, 2014,14(6):1114-1128.DOI: 10.1111/1755-0998.12259.

https://doi.org/10.1111/1755-0998.12259

https://www.ncbi.nlm.nih.gov/pubmed/24690386

本文引用 [1] 摘要

Fruit flies in the family Tephritidae are the economically important pests that have many species complexes. DNA barcoding has gradually been verified as an effective tool for identifying species in a wide range of taxonomic groups, and there are several publications on rapid and accurate identification of fruit flies based on this technique; however, comprehensive analyses of large and new taxa for the effectiveness of DNA barcoding for fruit flies identification have been rare. In this study, we evaluated the COI barcode sequences for the diagnosis of fruit flies using 1426 sequences for 73 species of Bactrocera distributed worldwide. Tree-based [neighbour-joining (NJ)]; distance-based, such as Best Match (BM), Best Close Match (BCM) and Minimum Distance (MD); and character-based methods were used to evaluate the barcoding success rates obtained with maintaining the species complex in the data set, treating a species complex as a single taxon unit, and removing the species complex. Our results indicate that the average divergence between species was 14.04% (0.00-25.16%), whereas within a species this was 0.81% (0.00-9.71%); the existence of species complexes largely reduced the barcoding success for Tephritidae, for example relatively low success rates (74.4% based on BM and BCM and 84.8% based on MD) were obtained when the sequences from species complexes were included in the analysis, whereas significantly higher success rates were achieved if the species complexes were treated as a single taxon or removed from the data set - BM (98.9%), BCM (98.5%) and MD (97.5%), or BM (98.1%), BCM (97.4%) and MD (98.2%).

[29]	ORTMAN B D, BUCKLIN A, PAGÈS F, et al. DNA Barcoding the Medusozoa using mtCOI[J]. Deep Sea Res Part II:Top Stud Oceanogr, 2010,57(24/25/26):2148-2156.DOI: 10.1016/j.dsr2.2010.09.017. 本文引用 [1]